Scientists solve CRISPR’s ‘Energizer bunny’ problem

olecule by molecule, the transformational genome-editing technology called CRISPR-Cas9 is getting so many upgrades so quickly it’s like scientists are changing a flip phone into a Galaxy S7 overnight. On Wednesday, scientists unveiled two more improvements that could speed the development of drugs and increase the chance of any CRISPR-based gene therapies succeeding.

“It’s remarkable science and unquestionably spectacular work,” said Eric Kmiec, director of the gene-editing program at Delaware’s Christiana Care Health System, who was not involved in the new study. “But the field is moving so quickly, I’m concerned that we might be doing things just because they’re really cool.”

The seemingly constant advances also may turn the bitter patent battle over CRISPR into a historical footnote. “If something else becomes the fundamental technology,” said Jacob Sherkow of New York Law School, then the original CRISPR patents — awarded to the Broad Institute and challenged by the University of California — “might be worth less than people envisioned.”

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The latest advances, reported in Nature, come on the heels of last week’s announcement of a hack that allows CRISPR to change a single DNA “letter” into another without wreaking collateral damage on the genome.

CRISPR-Cas9 is a complex of several molecules that has been widely hailed as a precise genome-editing technology. But the precision refers to where one component, the enzyme Cas9, cuts a genome. In contrast, what happens after Cas9 cuts DNA is messy.

For one thing, the cell typically shoves random bits of DNA onto the broken ends of the double helix, deletes other bits, and generally inflicts what Harvard University biologist George Church has called “genetic vandalism.” For another, Cas9 is such an enthusiastic DNA cutter that it doesn’t settle for cleaving its target only once. “Cas9 is the Energizer bunny” of DNA-cutting molecules, said Kmiec. “It keeps cutting and cutting.”

It cuts the DNA and then the cell repairs it, said biologist Marc Tessier-Lavigne, the president of Rockefeller University and senior author of the new study. “Then it cuts again and the cell repairs it again. You get cutting and repairing and cutting and repairing until the [target site] is obliterated.” Because each cut-and-repair introduces unintended DNA changes — those chaotic insertions and deletions — “it’s hard to get only the intended edits.”

CRISPR-Cas9’s enthusiasm for cutting DNA has another consequence: It usually edits both copies of a gene, one from mom and one from dad. Until now, there has been no good way to edit only one copy and leave the other alone.

That might seem like a good problem to have: If CRISPR-based gene therapies ever arrive, presumably fixing both disease genes would be beneficial. But for basic research on what goes wrong in a disease, as well as on drugs to treat it, an edit that makes cells carry only one copy of a disease gene “is more valuable” than an edit that creates two, said Kmiec. A double-dose of a bad gene “could be so severe that nothing will help” treat the disorder. In contrast, cells with only one copy of a gene related to, say, Alzheimer’s disease or Parkinson’s, might be treatable, making them more useful for drug research.

In the new paper, Tessier-Lavigne’s team describe how they overcame both problems: stopping Cas9 from cutting again and again, and editing one but not both copies of a target gene.

For their experiments, largely conducted by postdoctoral fellow Dominik Paquet and graduate student Dylan Kwart, the scientists introduced mutations next to CRISPR’s target. As a result, CRISPR couldn’t find its way to its target a second time, so Cas9 stopped cutting an already-edited gene and the cell stopped shoving in random bits of DNA.

“It works beautifully,” Tessier-Lavigne said. With standard CRISPR-Cas9, 6 percent to 35 percent of the edits were clean, without random insertions and deletions due to Cas9’s repeated cuts, but with their upgrade, the percentage of clean edits rose twofold to tenfold.

They call their method “CORRECT” (consecutive re-guide or re-Cas steps to erase CRISPR/Cas-blocked targets) and say it allows “scarless genome editing.” Paquet, Kwart, and Tessier-Lavigne have filed a patent application on the work.

“Making precise edits has been a problem” for CRISPR, said biomedical engineer Kris Saha, an expert in gene-editing at the University of Wisconsin-Madison. “They present a very smart way to do it.”

The scientists, partly funded by the New York Stem Cell Foundation, used a similar approach to make CRISPR edit only one copy of a gene and leave the other alone. They discovered that how close to its target CRISPR-Cas9 made its cut determined whether the double helix would be edited. “Distance let us control whether a repair template is incorporated into one or both copies of a gene,” said Tessier-Lavigne.

If CRISPR ever succeeds in treating patients, there are probably few cases where only one copy of a disease gene rather than both should be edited. But for research into the causes of disease, being able to compare cells with zero, one, or two disease-causing mutations “could be illuminating,” Tessier-Lavigne said.

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The experiments were all done on cells growing in lab dishes, so it remains to be seen if CORRECT works even in a lab mouse. Also unclear is whether this approach to keeping Cas9 on a leash is better than using a different DNA-cutting enzyme, such as one CRISPR pioneer Feng Zhang and his colleagues discovered last year.

One concern scientists raised about CORRECT is that it inserts a lot of molecules into cells, three to 20 times the amount with traditional CRISPR, said Saha. More research will be needed to show whether that’s more than cells can tolerate.

Almost certainly, genome-editing will keep getting upgrades until, at some point, they eclipse the CRISPR system that the Broad and UC are battling over. An old principle in patent law, called the reverse doctrine of equivalents, holds that “if an improvement is so revolutionary, it’s like it was a new invention” and not a modification of an existing one, said Sherkow. “If so, it might deserve standalone patent protection” and would not infringe on the disputed CRISPR patents.

In that case, scientists who want to edit genomes could license only the improvement, not the Broad or UC patents, which would become historical curiosities.